Response of adult mouse uterus to early disruption of estrogen receptor-alpha signaling is influenced by Krüppel-like factor 9.

Inappropriate early exposure of the hormone-responsive uterus to estrogenic compounds is associated with increased risk for adult reproductive diseases including endometrial cancers. While the dysregulation of estrogen receptor-alpha (ESR1) signaling is well acknowledged to mediate early events in tumor initiation, mechanisms contributing to sustained ESR1 activity later in life and leading to induction of oncogenic pathways remain poorly understood. We had shown previously that the transcription factor Krüppel-like factor 9 (KLF9) represses ESR1 expression and activity in Ishikawa endometrial glandular epithelial cells. We hypothesized that KLF9 functions as a tumor suppressor, and that loss of its expression enhances ESR1 signaling. Here, we evaluated the contribution of KLF9 to early perturbations in uterine ESR1 signaling pathways elicited by the administration of synthetic estrogen diethylstilbestrol (DES) to wild-type (WT) and Klf9 null (KO) mice on postnatal days (PNDs) 1-5. Uterine tissues collected at PND84 were subjected to histological, immunological, and molecular analyses. Compared with WT mice, KO mice demonstrated larger endometrial glands and lower endometrial gland numbers; DES exposure exacerbated these differences. Loss of KLF9 expression resulted in increased glandular ESR1 immunoreactivity with DES, without effects on serum estradiol levels. Quantitative RT-PCR analyses indicated altered expression of uterine genes commonly dysregulated in endometrial cancers (Akt1, Mmp9, Slpi, and Tgfbeta1) and of those involved in growth regulation (Fos, Myc, Tert, and Syk), with loss of Klf9, alone or in concert with DES. Our data support a molecular network between KLF9 and ESR1 in the uterus, and suggest that silencing of KLF9 may contribute to endometrial dysfunctions initiated by aberrant estrogen action.

Estimates of inbreeding depression for serum insulin-like growth factor I concentrations, body weights, and body weight gains in Angus beef cattle divergently selected for serum insulin-like growth factor I concentration.

Data for the current study were obtained from a divergent selection experiment in which the selection criterion was the average serum IGF-I concentration of 3 postweaning blood samples collected from purebred Angus calves. Multiple trait derivative-free REML procedures were used to obtain estimates of inbreeding depression for IGF-I concentration and for BW and BW gains measured from birth to the conclusion of a 140-d postweaning performance test. Included in the analysis were 3,243 animals in the A(-1) matrix, 2,182 of which had valid records for IGF-I concentration. Over the course of the entire selection experiment, inbreeding of the calf averaged 3.3% (SD = 3.1%) and inbreeding of the dam averaged 1.8% (SD = 2.7%). Mean inbreeding levels at the end of the study were 6.82 +/- 0.38% and 4.20 +/- 0.36% for calves and dams, respectively. Annual rates of increase in inbreeding of calves and dams were 0.36 +/- 0.01 (P < 0.0001) and 0.25 +/- 0.01%/yr (P < 0.0001), respectively. Insulin-like growth factor I concentration at d 28 (IGF28), 42 (IGF42), and 56 (IGF56) of the 140-d postweaning test and mean IGF-I concentration decreased by 0.62 +/- 0.88, 1.86 +/- 0.96, 1.92 +/- 0.89, and 1.48 +/- 0.76 ng/mL per 1% increase in inbreeding of calf. Only the regression coefficient for IGF56 differed significantly from zero, although the regression coefficients for IGF42 and mean IGF-I approached significance (P < 0.10). Increases in inbreeding levels of the dams also tended to result in reduced IGF-I concentrations, although the regression coefficients were not significantly different from zero. Inbreeding of calf had highly significant negative effects on all BW and BW gain traits examined, except for birth weight, with regression coefficients ranging from -0.74 +/- 0.20 kg/% increase in calf inbreeding for postweaning BW gain to -1.68 +/- 0.33 kg/% increase in calf inbreeding for off-test BW. Inbreeding of dam had a significant negative effect on birth weight of progeny and tended to have a negative effect on postweaning BW gain (P < 0.10). Preweaning gain of the progeny and BW other than birth weight were not influenced by increases in dam inbreeding. Results indicate that reductions in serum IGF-I concentration due to inbreeding may contribute to the decline in BW and BW gains that is typically associated with increases in inbreeding within populations.

The emerging role of Krüppel-like factors in endocrine-responsive cancers of female reproductive tissues.

Krüppel-like factors (KLFs), of which there are currently 17 known protein members, belong to the specificity protein (Sp) family of transcription factors and are characterized by the presence of Cys(2)/His(2) zinc finger motifs in their carboxy-terminal domains that confer preferential binding to GC/GT-rich sequences in gene promoter and enhancer regions. While previously regarded to simply function as silencers of Sp1 transactivity, many KLFs are now shown to be relevant to human cancers by their newly identified abilities to mediate crosstalk with signaling pathways involved in the control of cell proliferation, apoptosis, migration, and differentiation. Several KLFs act as tumor suppressors and/or oncogenes under distinct cellular contexts, underscoring their prognostic potential for cancer survival and outcome. Recent studies suggest that a number of KLFs can influence steroid hormone signaling through transcriptional networks involving steroid hormone receptors and members of the nuclear receptor family of transcription factors. Since inappropriate sensitivity or resistance to steroid hormone actions underlies endocrine-related malignancies, we consider the intriguing possibility that dysregulation of expression and/or activity of KLF members is linked to the pathogenesis of endometrial and breast cancers. In this review, we focus on recently described mechanisms of actions of several KLFs (KLF4, KLF5, KLF6, and KLF9) in cancers of the mammary gland and uterus. We suggest that understanding the mode of actions of KLFs and their functional networks may lead to the development of novel therapeutics to improve current prospects for cancer prevention and cure.

Nuclear receptor co-regulator Krüppel-like factor 9 and prohibitin 2 expression in estrogen-induced epithelial cell proliferation in the mouse uterus.

Estrogen, acting through its cognate receptor estrogen receptor-alpha (ESR1), is a critical regulator of uterine endometrial epithelial proliferation. Although the dynamic communication between endometrial stromal (ST) and epithelial cells is considered to be an important component in this process, key molecular players in particular compartments remain poorly defined. Here, we used mice null for Krüppel-like factor 9 (KLF9) to evaluate the contribution of this nuclear protein in ST-epithelial interactions underlying proliferative effects of estrogen. We found that in ovariectomized mice administered estradiol-17beta (E(2)) for 24 h, Klf9 null mutation resulted in lack of E(2)-induced proliferative response in all endometrial compartments. We demonstrated a negative association between Klf9 expression and nuclear levels of ESR1 transcriptional corepressor prohibitin (PHB) 2 in uterine ST and epithelial cells of E(2)-treated wild-type (WT) and Klf9 null mice. In early pregnancy uteri of WT mice, the temporal pattern of Klf9 transcript levels was inversely associated with that of Phb2. Deletion of Klf9 up-regulated uterine Phb2 expression and increased PHB2 nuclear localization in endometrial ST and epithelial cells, with no effects on the expression of the related Phb1. In the human endometrial ST cell line treated with E(2) for 24 h, Klf9 siRNA targeting augmented PHB2 transcript and increased nuclear PHB2 protein levels, albeit this effect was not to the extent seen in vivo with Klf9 null mutants. Our findings suggest a novel mechanism for control of estrogen-induced luminal epithelial proliferation involving ST KLF9 regulation of paracrine factor(s) to repress epithelial expression of corepressor PHB2.

Genetic parameter estimates for serum insulin-like growth factor I concentrations, and body weight and weight gains in Angus beef cattle divergently selected for serum insulin-like growth factor I concentration.

Data for the current study were obtained from a divergent selection experiment in which the selection criterion was the average serum IGF-I concentrations of 3 postweaning blood samples collected from purebred Angus calves. Multiple-trait derivative-free REML procedures were used to obtain genetic parameter estimates for IGF-I concentrations and for BW and BW gains measured from birth to the conclusion of a 140-d postweaning performance test. Included in the analysis were 2,674 animals in the A(-1) matrix, 1,761 of which had valid records for IGF-I concentrations. Direct heritability estimates +/- SE for IGF-I concentration at d 28, 42, and 56 of the postweaning period and for mean IGF-I concentrations were 0.44 +/- 0.07, 0.51 +/- 0.08, 0.42 +/- 0.07, and 0.52 +/- 0.08, respectively. Heritability estimates for maternal genetic effects ranged from 0.10 +/- 0.05 to 0.20 +/- 0.06. The proportion of total phenotypic variance due to the maternal permanent environmental effect was essentially zero for all measures of IGF-I concentrations. Genetic correlations of IGF-I concentrations with weaning and post-weaning BW ranged from 0.07 +/- 0.12 to 0.32 +/- 0.11 and generally demonstrated an increasing trend during the postweaning period. Averaged across the various measures of IGF-I, the genetic correlation of IGF-I with preweaning gain was 0.14, whereas the genetic correlation with postweaning gain was 0.29. Genetic correlations between IGF-I and BW gain were positive during all time intervals, except between weaning and the beginning of the postweaning test and from d 84 to 112 of the postweaning period. Environmental and phenotypic correlations of IGF-I with BW and BW gains were generally positive, but small. These results indicate that postweaning serum IGF-I concentration is moderately to highly heritable and has small positive genetic, environmental, and phenotypic correlations with BW other than birth weight and with pre- and postweaning gain. Therefore, if IGF-I proves to be a biological indicator of an economically important trait (e.g., efficiency of feed use for growth) in beef cattle, it should be possible to rapidly change IGF-I concentrations via selection without significantly altering live weight or rate of gain.

Analysis of female reproductive traits in Angus beef cattle divergently selected for blood serum insulin-like growth factor I concentration.

Insulin-like growth factor-I (IGF-I) is an anabolic polypeptide involved in reproductive performance in several species. The objectives of this study were to determine relationships of pregnancy rate, and age of heifers at puberty and at first calving with serum IGF-I concentration in Angus beef cattle. Data were obtained from an ongoing divergent selection experiment for IGF-I concentration involving purebred Angus cows. The IGF-I concentrations measured at Days 28, 42, and 56 of the 140-day postweaning test are abbreviated as IGF28, IGF42, and IGF56, respectively. Pregnancy rate did not differ between high and low IGF-I line females (P=0.95; n=2618), but high line heifers tended to be 4.02+/-2.18 days younger (P=0.07; n=281) at first calving. Residual correlations of age of heifers at first calving (AFC) with IGF-I measurements were not significant. The linear and quadratic terms for regression of AFC on IGF-I concentrations were also non-significant. Contrast analysis showed no difference in age at puberty between the high and low IGF-I line heifers (5.3+/-6.4 days earlier in the high line; P=0.43; n=51). Residual correlations of age of heifers at puberty with IGF28, IGF42, IGF56, and mean IGF-I were -0.30 (P=0.03), -0. 22 (P=0.12), -0.35 (P=0.01), and -0.34 (P=0.01), respectively. The observed relationships between female reproductive traits and IGF-I concentration in Angus beef cattle suggest complex and multiple roles for IGF-I in reproduction.

The secretory leukocyte protease inhibitor gene is a target of epidermal growth factor receptor action in endometrial epithelial cells.

The over-expression of epidermal growth factor receptor (EGFR) and its ligands, epidermal growth factor (EGF) and transforming growth factor-alpha, is a common feature of epithelial carcinomas and correlates with neoplastic progression. Secretory leukocyte protease inhibitor (SLPI), a member of the Kazal superfamily of serine anti-proteases, induces proliferation and promotes malignancy of epithelial cells and is expressed at high levels in multiple tumor types. In the present study, we have demonstrated that EGF increases SLPI expression in the human endometrial epithelial cell line Ishikawa in a dose- and time-dependent manner. We have shown that this effect of EGF occurs, in part, at the level of the SLPI promoter and involves the MAP kinase signaling pathway. We have further shown that EGF promotion of cell proliferation, but not induction of cyclin D1 gene expression, involves SLPI. Our results suggest that the regulation of SLPI expression by EGFR ligand(s) may represent a 'feed-forward' mechanism by which the enhanced proliferative and migratory properties of EGFR over-expressing cancer cells are sustained. Increased SLPI expression is likely an important component of altered EGFR signaling in human tumors and may have significant therapeutic implications in cancer progression.

Estimation of (co)variance components for reproductive traits in Angus beef cattle divergently selected for blood serum IGF-I concentration.

The objective of this study was to obtain estimates of (co)variance components for reproductive traits and insulin-like growth factor-I (IGF-I) concentration. Data were from a divergent selection experiment for blood serum IGF-I concentration in Angus beef cattle. Numbers of observations for mean IGF-I concentration of three blood samples taken at d 28, 42, and 56 of the 140-d postweaning test, scrotal circumference (SC), percentage of motile sperm cells (PMSC), percentage of morphologically normal sperm cells (PNSC), age of heifers at first calving (AFC), and calving rate (CR) were 1,848, 825, 596, 765, 294, and 2,092, respectively. Total number of animals in the numerator relationship matrix, including base animals, was 2,864, of which 1,861 were inbred. Estimates of direct heritability for IGF-I concentration of three blood samples collected at d 28, 42, and 56 of the postweaning test and for mean IGF-I concentration were 0.43+/-0.08, 0.51+/-0.09, 0.41+/-0.08, and 0.50+/-0.08, respectively. Estimates of direct heritability for SC, PMSC, PNSC, AFC, and CR were 0.51+/-0.13, 0.08+/-0.12, 0.47+/-0.07, 0.26+/-0.28, and 0.11+/-0.05, respectively. With the exception of age at first calving, estimates of maternal heritability and proportion of phenotypic variance that were due to permanent environmental effects of the dams were smaller than 0.21. Observations for calving rate were entered as either 1 (if calved) or 100 (if not calved). Estimates of additive genetic correlations of mean IGF-I concentration with SC, PMSC, PNSC, AFC, and CR were 0.35+/-0.11, 0.43+/-0.32, 0.00+/-0.03, -0.14+/-0.33, and -0.41+/-0.16, respectively. Environmental and phenotypic correlations for all of the traits with IGF-I measurements were smaller than 0.23. These results suggest that selection for increased serum IGF-I concentration should result in increased scrotal circumference, percent motile sperm cells, and calving rate.

Evaluation of serum insulin-like growth factor binding proteins (IGFBP) in Angus cattle divergently selected for serum IGF-I concentration.

Postweaning serum insulin-like growth factor-I (IGF-I) concentrations and serum IGF binding proteins (IGFBP) were investigated in 68 (1992 Fall-born) and 84 (1999 Fall-born) Angus cattle selected for either high or low serum IGF-I concentrations since 1989. Relative serum levels of IGFBP were determined by [125I]IGF-I Western ligand blotting. IGFBP species of 38-42, 34, 30, and 24 kDa were identified. The 34 kDa species was identified as IGFBP-2 by immunoblot analysis. No significant line effects were observed for any of the IGFBP. In both 1992 and 1999, heifers had higher IGFBP-2 levels than bulls (P<0.0005). In 1992 calves, relative levels of the 38-42 and 24 kDa species were significantly correlated with serum IGF-I concentration. In 1999 calves, none of the IGFBP were correlated with serum IGF-I, although IGFBP-2 was negatively correlated with several measures of body weight. No significant line effects were observed for growth or serum IGF-I traits in 1992 calves. However, 1999 high line calves had higher serum IGF-I concentrations and body weights than low line calves (P<0.05). In both 1992 and 1999 calves, bulls had higher serum IGF-I concentrations and body weights than heifers (P<0.05). Thus, while selection for high versus low serum IGF-I concentrations has resulted in divergence between the selection lines and also in changes in body weights, it has not resulted in changes in serum IGFBP levels. Furthermore, circulating IGFBP-2 appears to be higher in heifers than in bulls, and also appears to be negatively correlated with body weights.

Genetic parameter estimates for serum insulin-like growth factor-I concentration and ultrasound measurements of backfat thickness and longissimus muscle area in Angus beef cattle.

A divergent selection experiment for serum IGF-I concentration began at the Eastern Ohio Resource Development Center in 1989 using 100 spring-calving (50 high line and 50 low line) and 100 fall-calving (50 high line and 50 low line) purebred Angus cows. Following weaning, bull and heifer calves were fed in drylot for a 140-d period. Real-time ultrasound measurements of backfat thickness and longissimus muscle area were taken on d 56 and 140 of the postweaning test. Only ultrasound data from calves born from fall 1995 through spring 1999 were included in the analysis. At the time of this study, IGF-I measurements were available for 1,521 bull and heifer calves, and ultrasound data were available for 636 bull and heifer calves. Data were analyzed by multiple-trait, derivative-free, restricted maximum likelihood methods. Estimates of direct heritability for IGF-I concentration at d 28, 42, and 56 of the postweaning period, and for mean IGF-I concentration were 0.26 +/- 0.07, 0.32 +/- 0.08, 0.26 +/- 0.07, and 0.32 +/- 0.08, respectively. Direct heritabilities for ultrasound estimates of backfat thickness ranged from 0.17 +/- 0.11 to 0.28 +/- 0.12, whereas direct heritabilities for longissimus muscle area ranged from 0.20 +/- 0.10 to 0.36 +/- 0.12, depending on the time of measurement and the covariate used for adjustment (age vs. weight). Direct genetic correlations of IGF-I concentrations with backfat thickness at d 56 and 140 and with longissiumus muscle area at d 56 and 140 averaged 0.02, 0.20, -0.08, and 0.23, respectively, when age was used as the covariate for both IGF-I and ultrasound measurements. Corresponding genetic correlations when age was used as the covariate for IGF-I and weight was used as the covariate for ultrasound measurements were 0.05, -0.07, -0.22, and -0.04, respectively. Therefore, the positive associations of serum IGF-I concentration with backfat thickness and longissimus muscle area at d 140 seem to have been partially mediated by weight. Results of this study do not indicate strong associations of serum IGF-I concentration with fat thickness or muscling of bulls and heifers during the postweaning feedlot period.

Association of single nucleotide polymorphisms in the growth hormone and growth hormone receptor genes with blood serum insulin-like growth factor I concentration and growth traits in Angus cattle.

This study was conducted to identify polymorphisms in the promoter and coding regions of the bovine growth hormone and growth hormone receptor genes and to study association of polymorphisms identified in these genes with growth traits and serum insulin-like growth factor-I (IGF-I) concentration. The denaturing gradient gel electrophoresis method and sequencing were utilized to identify three new single nucleotide polymorphisms in the promoter region of the growth hormone gene in Angus cattle. Polymerase chain reaction-based restriction fragment length polymorphism procedures were developed for rapid determination of the single nucleotide polymorphism genotypes in the growth hormone and the growth hormone receptor genes among Angus calves from lines divergently selected for high or low blood serum IGF-I concentration. The IGF-I concentration and growth traits were analyzed using animal models. The single nucleotide polymorphism in the promoter region of the growth hormone receptor gene was associated with serum IGF-I concentration on d 42 of the postweaning test and with mean IGF-I concentration. The associated effects of the markers need to be verified in other populations.

Insulin-like growth factor-I and insulin-like growth factor binding protein-2 and -5 in equine seminal plasma: association with sperm characteristics and fertility.

The objectives of this study were 1) to determine whether insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding proteins (IGFBPs) were present in seminal plasma of stallions; 2) to compare semen parameters (IGF proteins, sperm numbers, morphology, and motility) from stallions at sexual rest (SR) and when sexually active (SA); 3) to compare semen parameters between stallions with high and low seminal plasma IGF-I concentrations; and 4) to examine the relationship between seminal plasma IGF-I concentrations and fertility parameters of stallions. Ejaculates were collected from stallions at SR (n = 51) and SA (n = 46). Concentrations of IGF-I and IGFBP-2 in seminal plasma samples were determined by radioimmunoassay. Presence of IGFBPs in equine seminal plasma was verified using immunoprecipitation and Western ligand blot procedures. IGF-I, IGFBP-2, and IGFBP-5 were present in equine seminal plasma. Concentrations of IGF-I, IGF-I/protein, total IGF-I, IGFBP-2, IGFBP-2/protein, and total IGFBP-2 were not significantly different (P > or = 0.13) in seminal plasma between stallions at either SR or SA. At SR, stallions with higher seminal plasma IGF-I had more total IGFBP-2 per ejaculate (P < 0.01), more morphologically normal sperm (P = 0.05), and higher first-cycle pregnancy rates (P = 0.02). At SA, stallions with higher seminal plasma IGF-I had fewer cycles per pregnancy (P = 0.02). An association of seminal plasma IGF-I concentration with sperm motility, sperm morphology, and pregnancy rates in bred mares suggests that IGF-I may play a role in sperm function.

Increased expression of the Zn-finger transcription factor BTEB1 in human endometrial cells is correlated with distinct cell phenotype, gene expression patterns, and proliferative responsiveness to serum and TGF-beta1.

Basic transcription element binding (BTEB, also designated BTEB1) protein is a member of the Sp-family of GC-box binding transcription factors that exhibit distinct patterns of expression in many cell types and tissues. A role for BTEB1 in the regulation of cell growth and gene transcription has been invoked, but little is known about the molecular mechanisms underlying these activities. The present study examined the functional consequences of high and low BTEB1 expression in the human endometrial carcinoma cell line Hec-1-A, by deriving stable clonal lines that expressed sense (S) and anti-sense (As) rat BTEB1 constructs. Clonal S lines, with BTEB1 mRNA and protein levels higher than in corresponding parent (N) and As lines, displayed enhanced DNA synthesis upon 3[H]-thymidine incorporation, in serum-containing but not in serum-free medium, and increased cell cycle kinetics, concomitant with the induction in expression of the genes for the cell cycle-associated components cyclin D1, PCNA, cyclin-dependent kinase (Cdk) inhibitor p21, and Cdk2. Compared to N and As lines, S lines also had diminished ability to grow in multi-layers and exhibited increased mRNA levels for plasminogen activator inhibitor-1 (PAI-1), secretory leukocyte protease inhibitor (SLPI), and tissue inhibitor of metalloproteinases (TIMP)-2. In serum-free medium, S, but not N nor As lines, had enhanced DNA synthesis with transforming growth factor (TGF)-beta1, albeit all lines demonstrated similar responses to insulin-like growth factor-I and to epidermal growth factor, respectively. The higher DNA synthesis in S relative to N and As, lines upon exogenous TGF-beta1 addition, was observed in concert with increased expression of cyclins D1 and E and p21, genes. Moreover, S and As lines had increased mRNA levels for TIMP-1, TIMP-2, PAI-1, and beta-catenin, and diminished SLPI, and to a lesser extent, Cdk4 mRNA levels, with TGF-beta1 treatment. These results suggest that BTEB1 may mediate cell growth, in part, by modulating gene expression levels of distinct cell cycle and growth-associated proteins. The correlation between serum- and TGF-beta1 induction of DNA synthesis with increased BTEB1 expression further suggests that BTEB1 may constitute an important downstream regulatory component of various signaling pathways utilized by serum-associated and other growth factors in endometrial epithelial cells.

Association of a genetic marker with blood serum insulin-like growth factor-I concentration and growth traits in Angus cattle.

The objective of this research was to evaluate a biallelic genetic marker identified in the first promoter region of the bovine IGF-I gene. The point mutation was identified as a T-to-C transition by sequencing the polymorphic fragments. A PCR-RFLP procedure was developed for determining the marker genotypes. Marker genotypes were determined for 760 Angus calves from divergent lines that were created by selection for high or low serum IGF-I concentration (allele A: 63.9%, B: 36.1%). Data were analyzed using the multiple-trait derivative-free restricted maximum likelihood computer programs with animal models. The full animal model included fixed effects of marker genotype, birth year, season of birth, sex, age of dam, and selection line; random effects of animal, maternal genetic, and maternal permanent environmental effects; and a covariate for age of calf. Traits analyzed included blood serum IGF-I concentrations on d 28, 42, and 56 of the postweaning test, mean IGF-I concentration, birth weight, weaning weight, on-test weight, off-test weight, off-test hip height, postweaning gain, and weight gain during the 20-d period immediately after weaning. Results from the analysis across selection lines showed a significant association of the BB genotype with higher weight gain during the first 20 d after weaning and a slight dominance effect of the marker on postweaning gain. Analysis within the low IGF-I line also showed a significant association of the BB genotype with higher weight gain during the first 20 d after weaning and with on-test weight, although analysis within the high IGF-I line did not show any significant association. The associated effects of the marker need to be verified in other cattle populations.

Polyamine- and insulin-like growth factor-I-mediated proliferation of porcine uterine endometrial cells: a potential role for spermidine/spermine N(1)-acetyltransferase during peri-implantation.

Insulin-like growth factor-I (IGF-I) and the polyamine catabolic enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) are progesterone-regulated genes with maximal expression at peri-implantation in the porcine uterine endometrium. However, while IGF-I stimulates cell proliferation, SSAT, by acetylating the naturally occurring polyamines (PA) spermine (SPM) and spermidine (SPD), typically functions as a cell growth inhibitor. The present study examined the functional relationships of IGF-I, SSAT, and PA in the control of endometrial cell proliferation. Northern blot analysis indicated that SSAT mRNA levels change with distinct pregnancy stages, in contrast to those for the PA biosynthetic enzyme ornithine decarboxylase (ODC). Primary cultures of luminal and glandular epithelial (LE, GE) and stromal (ST) cells isolated from Day 12 pregnant pig endometrium had IGF-I mRNA levels for ST > LE > GE cells. The mRNA levels for SSAT and ODC were transiently diminished by IGF-I treatment, but only in GE cells. By contrast, SPM and SPD increased SSAT mRNA levels in GE and ST cells, but increased ODC mRNA levels only in GE cells. IGF-I, putrescine (PUT), and SPM individually increased cellular DNA synthesis as measured by tritiated thymidine incorporation in GE and ST cells, while SPD had an effect only in ST cells. IGF-I enhanced the proliferative effect of each PA in GE cells, but only of SPD in ST cells. The mitogen-activated protein kinase inhibitor, PD98059, inhibited the induction by SPM of GE cell DNA synthesis but not that of IGF-I. Wortmannin, a phosphatidylinositol-3-kinase inhibitor had no effect on either IGF-I or SPM induction of GE cell DNA synthesis. The relative concentrations of SPM, SPD, and PUT in uterine luminal fluids differed, with the levels for each PA higher at pregnancy Day 12 than at 11.5. These results suggest that IGF-I and PA act through distinct signaling pathways to mediate cell-type-specific growth of early pregnancy pig uterine endometrium. Further, SSAT, through its control of intracellular PA levels, likely plays a modulatory role in the establishment of an optimal uterine environment for successful embryo attachment.

Molecular cloning of the porcine acid-labile subunit (ALS) of the insulin-like growth factor-binding protein complex and detection of ALS gene expression in hepatic and non-hepatic tissues.

The acid-labile subunit (ALS) is an approximately 85 kDa N-glycoprotein that is known primarily as a component of the systemic insulin-like growth factor-binding protein (IGFBP) complex. We have amplified, using a PCR, three overlapping porcine ALS genomic DNA fragments that together encode the distal region of the signal peptide through to the COOH-terminus. The compiled sequence of 1775 nucleotides of the three overlapping DNAs and the deduced amino acid sequence of the mature porcine ALS (pALS) protein exhibited 84/81%, 79/77%, 79/78% and 84/79% identities with respect to those of the human, the rat, the mouse and the baboon respectively. Four conserved cysteine residues in the NH(2)-terminal domain and 20 leucine-rich repeats in the central domain also were identified at identical positions in the porcine ALS. By using Northern blot analysis, with a genomic DNA fragment as the probe, it was determined that a 2.2 kb ALS mRNA was induced in the liver during the late fetal stage, and hepatic ALS mRNA abundance was increased post-natally. Moreover, hepatic ALS mRNA abundance was increased by daily injection of porcine somatotropin (100 microg/kg body weight) in cross-bred market pigs each weighing approximately 100 kg. The ALS mRNA was not detected by Northern analysis in any non-hepatic tissue examined. However, results of a more sensitive solution hybridization/RNAse protection assay indicated that low levels of ALS mRNA were also present in adult muscle, spleen, ovary and uterus, but not in lung, kidney, oviduct and placenta. Taken together, the present results suggest that although liver is the primary organ that expresses the ALS gene under somatotropin stimulation, some non-hepatic tissues also express the gene at low levels in the pig.

Genetic parameter estimates for serum insulin-like growth factor-I concentration and carcass traits in Angus beef cattle.

Divergent selection for serum insulin-like growth factor-I (IGF-I) concentration began at the Eastern Ohio Resource Development Center (EORDC) in 1989 using 100 spring-calving (50 high line and 50 low line) and 100 fall-calving (50 high line and 50 low line) purebred Angus cows. Following weaning, bull and heifer calves were fed in drylot for a 140-d postweaning period. At the conclusion of the postweaning test, bulls not selected for breeding were slaughtered and carcass data were collected at a commercial abbatoir. At the time of this analysis, IGF-I measurements were available for 1,283 bull and heifer calves, and carcass data were available for 452 bulls. A set of multiple-trait, derivative-free, restricted maximum likelihood (MTDFREML) computer programs were used for data analysis. Estimates of direct heritability for IGF-I concentration at d 28, 42, and 56 of the postweaning period, and for mean IGF-I concentration were .32, .59, .31, and .42, respectively. Direct heritabilities for carcass traits ranged from .27 to 1.0, .26 to 1.0, and .23 to 1.0 when the age-, fat-, and weight-constant end points, respectively, were used, with marbling score having the smallest heritability and longissimus muscle area having the highest heritability in each case. Maternal heritability and the proportion of phenotypic variance due to permanent environmental effect of dam generally were < or = .21 for IGF-I concentrations and for carcass traits other than longissimus muscle area. Additive genetic correlations of IGF-I concentrations with backfat thickness, longissimus muscle area, hot carcass weight, marbling score, quality grade, and yield grade averaged -.26, .19, -.04, -.53, -.45, and -.27, respectively, when carcass data were adjusted to an age-constant end point. Bulls with lower IGF-I concentrations had higher marbling scores and quality grades, but also had higher backfat thickness and yield grades regardless of the slaughter end point. Serum IGF-I concentration may be a useful selection criterion when efforts are directed toward improvement of marbling scores and quality grades of beef cattle.

Pregnancy-dependent expression of leukaemia inhibitory factor (LIF), LIF receptor-beta and interleukin-6 (IL-6) messenger ribonucleic acids in the porcine female reproductive tract.

Leukaemia inhibitory factor (LIF) and interleukin-6 (IL-6) are candidate embryo-maternal signalling molecules which are present within the uterine luminal micro-environment. We examined the relative expression of the mRNAs encoding LIF and IL-6, as well as the LIF-binding subunit (LIFR-beta) of the LIF receptor and, as a potential downstream cytokine-responsive gene, beta(2)-microglobulin (beta(2)m), in porcine peri-implantation conceptuses, and in placenta and endometrium during early and mid-pregnancy. Peri-implantation spherical and filamentous conceptuses expressed LIFR-beta and beta(2)m mRNAs with no LIF mRNA present. Rapid development in days 11/12 spherical conceptuses to the filamentous stage was accompanied by transiently increased IL-6 gene expression. The corresponding endometrium, in contrast, expressed LIF in addition to these other mRNAs. LIFR-beta, IL-6 and beta(2)m, but not LIF mRNAs, were expressed in the Jag-1 cell line, an in vitro model for porcine day 14 trophoblast. The greatest steady-state amounts of LIF, LIFR-beta and IL-6 mRNAs in both the endometrium and placenta were evident at the post-implantation stages (days 30 and 60>day 18 of pregnancy). Treatment of porcine endometrial explants with human recombinant (hr)LIF or hrIL-6 resulted in no change in, or diminished, the presence of endometrial beta(2)m mRNA, respectively. Addition of LIF to peri-implantation conceptus explant cultures, in contrast, induced beta(2)m mRNA synthesis. These results highlight the potential importance of both the endometrium and placenta as sources, as well as targets, of these cytokines throughout pregnancy. Cytokine modulation of beta(2)m, a known in vitro mitogen, may constitute one mechanism for local control of trophoblast and endometrial proliferation.

Multiple isoforms of porcine aromatase are encoded by three distinct genes.

Cytochrome P450 aromatase, a product of the CYP 19 gene and the terminal enzyme in the estrogen biosynthetic pathway, is synthesized by the ovary, endometrium, placenta, and peri-implantation embryos in the pig and other mammals, albeit to varying levels, implying its functional role(s) in pregnancy events. The aromatase produced by the pig tissues exists as three distinct isoforms (type I - ovary, type II - placenta, and type III - embryo), with presumed differences in substrate specificities, expression levels, activity, and mode of regulation. In order to delineate the molecular mechanisms whereby estrogen synthesis is regulated in these diverse tissues, the present study examined if these aromatase isoforms represent products of multiple genes or of a single gene via complex splicing mechanisms. Porcine genomic DNA from a single animal was used as a template in the polymerase chain reaction (PCR) to amplify isoform-specific sequences corresponding to exons 4 and 7, respectively. Nucleotide sequence analysis of the generated fragments revealed the presence of only clones corresponding to the three known aromatase types. Screening a porcine Bacterial Artificial Chromosome (BAC) library for aromatase gene by PCR yielded a single clone approximately 80 kb in length. Southern blot analysis, using probes specific for exons 1A-1B, 2-3, 4-9, and 10 sequences indicated that the BAC genomic clone contains the entirety of the coding exons as well as the proximal promoter region. Sequence analysis of the fragment generated with exon 4 primers determined that this BAC clone contains only the type II gene. The presence and relative orientation of the untranslated 5'- exons 1A and 1B, previously demonstrated for the type III isoform were evaluated in the BAC clone and genomic DNA by PCR. The 265 bp fragment generated from both PCR reactions was confirmed by sequence analysis to contain exons 1A and 1B that are located contiguous to each other and separated by only three bp. A diagnostic procedure for typing aromatase isoforms was developed, based on the presence of specific restriction sites within isoform-specific exons. The use of this protocol confirmed the existence of only three aromatase isoforms in the porcine genome and indicated changes in aromatase types expressed by the uterine endometrium as a function of pregnancy stage. The presence of distinct genes encoding each of the aromatase isoform predicts important differences in the mechanisms underlying the molecular evolution and regulation of porcine aromatase, unique from those of other mammals, and suggests a critical role for P450 aromatase steroidal products in uterine functions related to pregnancy events.

Expression and regulatory function of the transcription factor Sp1 in the uterine endometrium at early pregnancy: implications for epithelial phenotype.

The uterus during early pregnancy synthesizes a complex array of signaling molecules with specific spatial and temporal modes of expression and which are critical for embryo implantation and subsequent development. The mechanism(s) underlying the differential pattern of synthesis of these pregnancy-associated proteins is not understood very well. The present study evaluated the expression and trans-activation potential of the transcription factor Sp1 in the early pregnancy porcine endometrium to determine its temporal and functional association with the endometrial epithelial-specific genes encoding the transplacental iron-transport protein uteroferrin (UF) and an Sp-family member, basic transcription element-binding (BTEB) protein. Two identical Sp1 clones (717 bp) were isolated from a porcine endometrial cDNA library by polymerase chain reaction (PCR). The nucleotide sequence of these clones encodes a partial protein sequence of 238 amino acids encompassing the Zn-finger region and had significant identities with the corresponding regions in the rat and human proteins. By using a specific antibody raised against human Sp1, porcine endometrial Sp1 was found to exhibit a molecular weight of 110 kDa, was localized predominantly in the nuclei of glandular and luminal epithelial cells, and appeared to exist as a phosphorylated protein. Northern blot analysis demonstrated three distinct size transcripts of approximately 3.5, 5, and 8 kb for endometrial Sp1. The expression of Sp1 mRNA and protein, determined by RT-PCR and by its ability to bind Sp1 consensus motif in gel mobility shift assays, respectively, overlapped with, but did not parallel that of UF mRNA during early pregnancy. The effect of increased Sp1 expression on UF gene promoter activity was examined using a human Sp1 expression vector that was transiently transfected into primary cultures of pig endometrial glandular epithelial cells. Sp1 increased (P < 0.05) the promoter activities of various UF promoter-Luciferase reporter constructs by 2 to 4-fold, over those transfected with empty expression vector. Co-transfection of a BTEB expression vector with the Sp1 expression vector modified the effect of Sp1 on UF promoter activity in the shortest construct. These results suggest that Sp1 mediates the regulation of endometrial epithelial gene expression during pregnancy, and that this function is likely altered in vivo by co-expression of other family members, including BTEB.